We have discovered a number of novel features of the ability of the facultative intracellular bacterium, L. monocytogenes to induce potent immunity against E7 expressing tumors, which may be generally applicable to other cancer approaches and to the measurement of immune components that are indicative of good tumor immunotherapy. Thus, in constructing the L. monocytogenes recombinants that express tumor specific antigens we have found that the expression of the antigen as a fusion protein with the listerial protein listeriolysin O (Lm-LLO-E7) results in a much more potent tumor immunotherapeutic than if the antigen is expressed alone (Lm-E7), even when delivered by a vaccinia viral vector or as a plasmid DMA vaccine. This appears to be due to a the induction of CD4+ CD25+ regulatory T cells by Lm-E7 coupled with an improved ability to induce tumor-homing CD8+ T cells by Lm-LLO-E7. We request funding to continue to explore these findings in an HPV-E7 system consisting of mouse tumors, naturally immortalized by HPV, transplanted into either syngeneic wild type mice, or syngeneic mice transgenic for the E7 gene, to test for the ability of the therapeutic approaches to break tolerance. For specific Aim 1, we hypothesize that the efficacy of E7 based immunogens in inducing the regression of established cancer is related to the type of T cell immunity induced by Lm-E7 versus Lm-LLO-E7 and will focus on (a) how T regulatory cells can be induced by a "vaccine" and (b) the factors in the tumor milieu that control T cell tumor homing and penetration. In Specific aim 2 we hypothesize that the enhanced immunity conferred by delivering a tumor antigen fused to LLO is partly due to the presence of a PEST amino acid sequence in the LLO protein and also with the ability of LLO to mature dendritic cells and increase surface expression of immunostimulatory molecules. In this specific aim we will explore the mechanism by which this protein virulence factor is capable of improving the immunogenicity of tumor-associated antigens. Finally in Specific Aim 3, we will examine the ability of Lm- LLO-E7 to break tolerance in E7-transgeic mice where the HPV-16 E7 gene is a self-antigen expressed in thyroid follicular cells and develop malignant thyroid tumors. In this specific aim we hypothesize that Lm- LLO-E7 will break tolerance in this mouse model and we will explore the mechanisms by which it does so. [unreadable] [unreadable] [unreadable]